PROJECT SUMMARY Fracture healing is a complex bone healing process regulated by various classes of cells and signaling mechanisms. Of these, Wnt signaling has long been known to be critically important in directing the differentiation of mesenchymal stem cells (MSCs) into osteoblasts during bone healing. However, the source of these Wnt ligands is not known. Recently, a body of literature has arisen finding both beneficial and detrimental effects of macrophage-derived Wnt ligands on the regeneration of other tissues (liver, kidney, heart, and intestine), often by their modulation of progenitor cell behavior. As macrophages have been well-characterized as orchestrators of healing, their use of Wnt signaling to regulate tissue regeneration is unsurprising. However, no studies have yet identified the contribution of macrophages to Wnt signaling during fracture healing. Interestingly, recent unpublished data suggests that macrophages represent a major source of Wnt ligands during bone healing and underscore the importance of macrophages in driving this process. In the work proposed here, the expression of Wnt ligands by macrophages will first be measured, as well as the cells targeted by Wnt ligands, during fracture healing. Then, Wls, the gene encoding a necessary Wnt ligand trafficking protein, will be deleted in Csf1r- expressing cells (monocytes and macrophages), inhibiting the secretion of all Wnt ligands by these cells after induction of Cre recombinase with tamoxifen. Following deletion of macrophage Wls, the consequences of loss of macrophage-derived Wnt ligands will be characterized during the inflammatory, soft callus, and hard callus phases of healing. Appropriate transition through these phases, and ultimately successful healing, will be evaluated using qPCR, flow cytometry, micro-computed tomography, and histology. Finally, the mechanism of Wnt trafficking to MSCs, and the consequences of their effects on MSC behavior, will be evaluated through in vitro co-culture experiments. The studies defined in this proposal will elucidate the importance of macrophage Wnt ligands on bone healing and potentially identify Wnt signaling as a targetable pathway to enhance successful osseous healing.